Our previous studies led us to propose that a sex determination regulatory gene, fruitless (fru), is responsible for building the potential for male sexual behaviors into the CNS in D. melanogaster, and that the neural circuits underlying other complex innate behaviors are also likely constructed by the action of specific regulatory hierarchies (Baker, Taylor and Hall, 2001). Recently we showed that the FruM proteins are both necessary and sufficient to specify the potential for male courtship behavior. Moreover, the FruM proteins are expressed exclusively in subsets of the CNS and the primary sensory neurons of all sensory systems implicated in courtship. Expression is maximal about two days into the pupal period, when ca. 2000 cells (~2% of neurons) express FruM proteins. Strikingly, these neurons are dedicated to sexual behaviors, as inactivating them has no discernable effects other than on sexual behaviors. The findings that FruM proteins are expressed in only a small portion of the nervous system that is dedicated to sexual behavior, and are necessary and sufficient for nearly all aspects of sexual behavior are provocative and pleasing. They suggest FruM provides a handle for dissecting the developmental, genetic, molecular, and neuronal bases of male courtship behavior. We believe the key to gaining an understanding of how (1) the potential for a complex behavior is built into the nervous system, and (2) the neurons subserving male courtship behavior function together to insure the ordered manifestation of the events comprising this behavior, will be to focus on the groups of neurons in which the FruM proteins are expressed. On-the-one-hand we address how the FruM transcription factors shape the anatomical and molecular characteristics of these neurons. On-the-other hand we address the roles of individual groups of these neurons in the complex set of behaviors that comprise male courtship. Thus this grant focuses on the roles that these neurons play in adult male sexual behavior, and the fru-dependent characteristics that distinguish them. A long-term goal is to elucidate the structure of the FruM-specified courtship circuitry and how it functions. Central to our approach is the development of fru-based genetic tools that permit the manipulation of FruM-expressing neurons without affecting other neurons. Such constructs allow visualization of the nuclei of FruM-expressing neurons and their projections, silencing of these neurons, changing the sex of these cells from male to female or from female to male, suppression of FruM synthesis in targeted neurons. Thus we can functionally manipulate a discrete group of FruM neurons and behaviorally assess its effects on the execution of male courtship. We can also use these tools to identify the neuroanatomical and molecular characteristics of neurons that are specified by FruM. These studies will provide a model for how the circuitries underlying other innate behaviors are built and function. PUBLIC HEALTH RELEVANCE Our research promises to yield an unprecedented, detailed view of the molecular, cellular and behavioral basis for gender differences in the model genetic organism Drosophila melanogaster. In addition, these studies will provide a model for how the potential for other innate behaviors are genetically specified in the nervous system during development.